Characterizing Adeno-Associated Virus Capsids with both Denaturing and Intact Analysis Methods

Abstract

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein sequences, with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivty for viral protein (VP) subunits due to their sequence similiaries, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainity in distinguishing empty and filled capsids. To address these challenges, we combine two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids and then liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were used for capsid denaturing measurements. This multi-method combination was applied to 3 AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.

Instrumentation at the Leading Edge of Proteomics

“…209,210,705−707 The coming years will be exciting times for CDMS and intact protein analysis, especially as it has (so far) been commercialized as Direct Mass Technology on the newest generation Q-Orbitrap platforms. 207,208,704,708 Structural Proteomics. Deriving the protein structure from MS data has long been a goal of the proteomics community, and modern instrumentation has helped make it a reality.…”

“…CDMS is a single-ion detection strategy used to simultaneously measure charge (via induction of an image current) and m/z (traditionally via TOF). , In this way, CDMS provides mass measurements of large biomolecules (beyond MDa masses) that typically cannot be assigned a charge state in ESI-FTMS because resolution is not sufficient for isotopic resolution . The Kelleher and Heck groups recently demonstrated that individual ion CDMS measurements can be made within commercial Orbitrap systems rather than requiring custom MS hardware often used for CDMS. − ,, Orbitrap CDMS is particularly promising for DNA-containing viral capsids, glycoproteins, protein complexes, and amyloid protein aggregates, and it also boosts performance of top-down MS via improvements to sensitivity and protein sequence coverage. ,,− The coming years will be exciting times for CDMS and intact protein analysis, especially as it has (so far) been commercialized as Direct Mass Technology on the newest generation Q-Orbitrap platforms. ,,, …”

Instrumentation at the leading edge of proteomics

“…[207][208][209][210]704,705 Orbitrap CDMS is particularly promising for DNA-containing viral capsids, glycoproteins, protein complexes, and amyloid protein aggregates, and it also boosts performance of top-down MS via improvements to sensitivity and protein sequence coverage. 209,210,[705][706][707] The coming years will be exciting times for CDMS and intact protein analysis, especially as it has (so far) been commercialized as Direct Mass Technology on the newest generation Q-Orbitrap platforms. 207,208,704,708 Structural proteomics Deriving protein structure from MS data has long been a goal of the proteomics community, and modern instrumentation has helped make it a reality.…”

Instrumentation at the leading edge of proteomics

“…[196][197][198][199]677,678 Orbitrap CDMS is particularly promising for DNA-containing viral capsids, glycoproteins, protein complexes, and amyloid protein aggregates, and it also boosts performance of top-down MS via improvements to sensitivity and protein sequence coverage. 198,199,[678][679][680] The coming years will be exciting times for CDMS and intact protein analysis, especially as it has (so far) been commercialized as Direct Mass Technology on the newest generation Q-Orbitrap platforms. 196,197,677,681 Structural proteomics Deriving protein structure from MS data has long been a goal of the proteomics community, and modern instrumentation has helped make it a reality.…”

“…198,199,[678][679][680] The coming years will be exciting times for CDMS and intact protein analysis, especially as it has (so far) been commercialized as Direct Mass Technology on the newest generation Q-Orbitrap platforms. 196,197,677,681 Structural proteomics Deriving protein structure from MS data has long been a goal of the proteomics community, and modern instrumentation has helped make it a reality. From the intact protein angle, native proteomics relies on non-denaturing solvents and ESI to study protein structure in the gas-phase.…”